Plasmodium falciparum (Pf) is the most lethal of the five species of malaria affecting 3.4 billion humans annually. The Pf life cycle consists of a single round of sexual and asexual replication in the mosquito gut and human liver, respectively, followed by multiple rounds of asexual replication in human red blood cells (RBCs). Proper transcriptional regulation at each stage is essential for the successful completion of the life cycle, for gametocytogenesis and for RBC invasion. Epigenetic enzymes are thought to play important and potentially essential roles in this regulation. The methylation state of histones at specific genomic loci, for example, controls the expression of stage-specific transcription factors such as AP2-G, of nutrient uptake channels including the Clag3 gene paralogs, and of mono-allelically expressed multi-gene families such as the highly virulent var genes that encode adhesion proteins involved in antigenic variation. These loci are kept transcriptionally repressed by trimethylation of lysine 9 on histone 3 (H3K9me3). Proper temporal activation of these loci or switching to a distinct gene form for antigenic variation requires the enzymatic removal of this H3K9me3 transcriptional silencing mark. Similarly, proper temporal repression of active genes marked by H3K4me3 such as transcriptionally active stage-specific genes or mono-allelic loci for gene switching, requires erasing the activating H3K4me3 mark. Demethylation of trimethylated histones such as H3K9me3 or H3K4me3 can only be carried out by Jumonji domain containing histone hydroxylases. No other proteins with this activity are known in biology to date. The Pf genome encodes three Jumonji hydroxylases: PfJmjC1, PfJmjC2 and PfJmj3. We propose that the aggregate activity of these enzymes is essential to Pf viability. We have a long standing interest in defining and pharmacologically modulating Jumonji enzyme function. Our Specific Aims here are: Aim 1: To characterize the anti-malaria mechanism of action of three potent & selective novel Jumonji demethylase inhibitors we have identified in an anti-malaria screen Aim 2: To determine the function of the three putative malaria Jumonji histone demethylases (focusing on PfJmj3 whose function has never been studied) This proposal will establish links between the molecular players driving malaria epigenetic pathways and events that cause pathology and immune evasion in humans, and will create a novel therapeutic pipeline of potential anti-malarial compounds targeting putative parasite Jumonji domain-containing epigenetic enzymes.